DNA interstrand crosslinking agents are highly deleterious lesions and potent mutagens to which humans are exposed from both exogenous and endogenous sources. In addition, this class of drugs, which include cyclophosamide, cis-platin, busulfan, and mitomycin C, are widely used as anti-cancer chemotherapeutics. Nevertheless, the cellular responses to these drugs in terms of both the cell cycle and DNA repair remain poorly understood. During the last grant cycle we were able to identify a number of novel proteins involved in the cellular ICL response. In addition, we have developed a number of new assays that have increased our ability to probe the mechanisms of ICL repair. There appears to be at least two distinguishable pathways of ICL repair in mammalian cells one of which occurs in G1/G0, and a second that is induced by stalled replication forks during S phase. The focus of this project will be on increasing our understanding of the various aspects of the S phase pathway of ICL repair. Specifically, we examine recruitment of repair and checkpoint proteins to ICLs using a novel laser microirradiation approach which can be used to crosslink psoralen to a defined subregion of the mammalian nucleus. Secondly, we will examine the mechanisms of fork collapse, and define proteins that are directly involved in ICL removal. Thirdly, we will investigate the role of candidate proteins in various stages of ICL repair processing. Fourthly, we will isolate stalled replication forks and using mass spectrometry we will identify proteins involved in repair and checkpoint functions that are recruited to these structures. Together the successful completion of these aims should greatly increase our understanding of the mechanisms by which ICLs are processed in mammalian cells, and thereby lead to potential new or enhanced chemotherapies for cancer.
This Project is part of a multicomponent Program Project with the theme of understanding the processing of complex DNA damage by mammalian cells. The significance to human health is to generate new knowledge and paradigms for modeling DNA repair of DNA interstrand crosslinks (ICLs), to improve therapy using ICL-inducing compounds, and to identify new therapeutic targets for cancer treatment.
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